Supplementary Materials for

Human OTULIN haploinsufficiency impairs cell-intrinsic immunity to

staphylococcal α-toxin

András N. Spaan et al.

Corresponding authors: András N. Spaan, a.n.spaan@umcutrecht.nl; Jean-Laurent Casanova, casanova@rockefeller.edu

Science 376, eabm6380 (2022)

DOI: 10.1126/science.abm6380

The PDF file includes:

Case Reports for the Patients Studied

Materials and Methods
Figs. S1 to S7
References

Other Supplementary Material for this manuscript includes the following:

Tables S1 to S4
MDAR Reproducibility Checklist
 Case reports for the patients studied
Patient A.II.5 was born to unrelated Dutch parents and had an unremarkable medical
history. He was vaccinated according to national guidelines. At the age of 19 years, and following
a seasonal influenza B virus infection, he was admitted to the intensive care unit (ICU) for bilateral
community-acquired necrotizing pneumonia. Methicillin-resistant S. aureus (MRSA) was isolated
from sputum, bronchoalveolar lavage and blood cultures. With the exception of leukocytosis
dominated by neutrophilic granulocytes, no abnormalities were found in routine leukocyte
differentiation tests upon admission to the ICU. The pneumonia was progressive, and the patient
developed an adult respiratory distress syndrome with septic shock. Despite antibiotic treatment
and supportive therapy, the patient died from multiple organ failure. The patient’s parents and
siblings were screened for MRSA carriage and all tested negative. The patient’s OTULIN variant
(p.D246V) was carried by his mother and both siblings. The mother, patient A.I.1, who is now 60
years old, had suffered from two episodes of community-acquired pneumonia, one of which
required admission to a general hospital ward for intravenous treatment with antibiotics. Routine
leukocyte differentiation tests in patient A.I.1 revealed no abnormalities. The sister, patient A.II.4,
is now 30 years old. She reported multiple episodes of mild upper respiratory tract infections and
one episode of pneumonia during childhood, none of which required hospitalization. Diagnostic
cellular immunophenotyping in patient A.II.4 revealed no abnormalities and normal levels of
immunoglobulins, with detectable titers of antibodies against various serotypes of Streptococcus
pneumoniae and Haemophilus influenzae type B. No complement deficiencies were detected, and
no defects were found in the oxidative burst response of neutrophilic granulocytes to PMA and
Escherichia coli. The brother, patient A.II.3, is now 25 years old. He reported no respiratory tract
infections, but self-reported unusually severe acne vulgaris as a teenager and several episodes of
impetigo.
Patient B.III.5, a 70-year-old Argentinian man, was born to non-consanguineous parents,
and suffered from recurrent episodes of necrotizing cellulitis accompanied by septic shock. His
first episode of necrotizing cellulitis occurred at the age of 14 years and he has since had four
additional episodes, affecting various part of his body, two of which required skin grafts. S. aureus
was repeatedly cultured from diagnostic samples obtained from the affected sites. The patient had
been admitted to the hospital on multiple occasions for the treatment of community-acquired
pneumonia with pneumosepsis. A dihydrorhodamine test revealed a normal oxidative burst in
neutrophilic granulocytes. Diagnostic cellular immunophenotyping revealed no abnormalities and
normal levels of immunoglobulins, with detectable titers of antibodies against various serotypes
of S. pneumoniae, measles virus, and rubella virus. No complement deficiencies were detected.
The patient’s OTULIN variant (p.E95X) was also carried by his brother (patient B.III.6) and his
son (patient B.IV.7). Patient B.III.6, now 72 years old, reported recurrent furunculosis requiring
surgical drainage. Patient B.IV.7, now 37 years old, reported multiple episodes of community-
acquired pneumonia, three of which required hospitalization for treatment with intravenous
antibiotics. Like his father, patient B.IV.7 had no immunological abnormalities on routine
diagnostic tests. The patient’s mother and maternal grandmother reportedly suffered from the same
infectious phenotype as patients B.III.6 and B.IV.7 during their lifetimes and died of non-
infectious causes at an advanced age.
Patient C.III.5, a boy born to unrelated Mexican parents, was vaccinated in accordance
with national guidelines. An exploratory laparotomy with appendectomy at the age of four years
was complicated by surgical wound infections, with abscess formation and episodes of abdominal
sepsis requiring admission to the intensive care unit. At eight years of age, following an episode

2
of community-acquired pneumonia, the patient developed spontaneous abscesses in the neck with
septic shock. Surgical debridement revealed extensive necrotic tissue and fascia involvement
necessitating skin graft transplantation. In the same year, the patient developed cellulitis of the leg,
requiring intravenous antibiotic therapy. At the age of 10 years, the patient was admitted to the
hospital for the drainage of multiple gluteal abscesses. The postoperative course was complicated
by septic shock and deep tissue infections of the surgical wounds requiring skin graft
transplantations. At the age of 16 years, an episode of community-acquired pneumonia was
complicated by empyema. Between the ages of 16 and 18 years, the patient required surgery for
the drainage of recurrent gluteal abscesses. Microbiological cultures revealed a polymicrobial
etiology, including S. aureus. A dihydrorhodamine test revealed that the oxidative burst was
normal in neutrophilic granulocytes. Repeated routine leukocyte differentiation tests revealed no
abnormalities, with normal levels of immunoglobulins and no complement deficiencies. At the age
of 18 years, the patient was diagnosed with recent human immunodeficiency virus infection, his
last negative result for infection with this virus having been obtained at the age of 16 years. The
patient subsequently refused further treatment for any of his conditions. At the age of 19 years,
following a prolonged period of wasting, the patient died. The patient’s OTULIN variant
(p.D268TfsX5) was also carried by his mother (patient C.II.3). The mother, now 50 years old,
suffered from non-inborn epilepsy of unknown etiology, and reported no particular infections of
the respiratory tract or skin.
Patient D.II.5 is an 11-year-old boy who was born to non-consanguineous French parents
and had suffered from recurrent severe necrotizing pneumonia, with intermittent recovery, since
the age of 1.5 years. Despite repeated attempts at diagnosis, no microbiological cause was
identified in analyses of bronchoalveolar lavage. Broad-spectrum antibiotics had no significant
impact on recovery, but salvage therapy with steroids resulted in clinical improvement. A
dihydrorhodamine test showed a normal oxidative burst in neutrophilic granulocytes. Diagnostic
cellular immunophenotyping revealed no abnormalities and normal levels of immunoglobulins,
with detectable titers of antibodies against various serotypes of S. pneumoniae, diphtheria and
tetanus toxins, and poliovirus. No complement deficiencies were detected. The patient’s OTULIN
variant (p.N341D) was also carried by his mother (patient D.I.1). The mother, now 35 years old,
reported no particular infections of the respiratory tract or skin.
Patient E.II.5, a monozygotic twin, is a 35-year-old woman who was born to non-
consanguineous Belgian parents. Following a cesarean section, the patient suffered extensive
necrosis of the skin surrounding the surgical wound. Her twin sister (patient E.II.4) suffered a
similar episode of skin necrosis following elective surgery for breast reconstruction. No
microbiological cause was identified in specimens taken from the affected wound areas.
Pathological examinations for both patients revealed numerous intra-epidermal neutrophils with
the formation of intra-epidermal pustules and vesicles. The dermis region was filled with abscesses
containing neutrophils and debris. These findings were suggestive of pyoderma gangrenosum.
Diagnostic cellular immunophenotyping revealed no abnormalities and normal levels of
immunoglobulins. Lymphocyte mitogen stimulation tests were also normal and a
dihydrorhodamine test showed a normal oxidative burst in neutrophilic granulocytes. The patients’
OTULIN variant (p.P254S), was also carried by their father (patient E.I.2) and sister (patient
E.II.3). Patients E.I.2 and E.II.3 reported no particular infections of the respiratory tract or skin, or
complications of wound healing following surgery.
Patient F.II.3, a 46-year-old French man with non-consanguineous parents, had early
complicated Crohn’s disease beginning at the age of 10 years and requiring extensive colorectal

3
surgery. At the age of 42 years, during a relapse of his Crohn’s disease that was poorly controlled
by biotherapies, the patient presented severe hidradenitis suppurativa (stage 3 according to the
Hurley grading system) affecting his axillae, anal cleft, pubis, and inguinal-genital area. Based on
a suspected polymicrobial origin of the skin lesions, with the involvement of staphylococci (107),
prolonged broad-spectrum intravenous antibiotic therapy was administered (108). Following this
antibiotic induction therapy, the patient was in remission for hidradenitis suppurativa and his
Crohn’s disease improved significantly. Routine leukocyte differentiation tests showed no
abnormalities, with normal levels of immunoglobulins. The patient’s relatives refused genetic
investigations into carriage of the patient’s OTULIN variant (p.R263Q).
The clinical phenotypes of the ORAS family kindreds carrying deleterious alleles in
heterozygosity were self-reported. Patient I.I.1, a 39-year-old Turkish woman (OTULIN variant
p.Y244C (32)), and patient J.I.2, an adult Iranian man (carrying an essential splicing site mutation
in the donor site of exon 6 (c.864+2 T>C) resulting in OTULIN variant p.W199-Q288del (31)),
both reported recurrent furunculosis occurring from adolescence. No specimens were obtained for
culture. Patient H.I.2, a 43-year-old Turkish man (OTULIN variant p.G174DfsX2 (32)), reported
recurrent episodes of community-acquired pneumonia during his childhood, one of which required
admission to a general hospital ward for treatment with intravenous antibiotics. The other carriers
of deleterious heterozygous OTULIN variants in these ORAS family kindreds (G.I.1, G.I.2, G.II.3,
H.I.1, I.I.2, I.II.3, K.I.1, K.I.2, K.II.3 (29-32)) reported no particular phenotype.
The clinical phenotypes of the individuals with 5p- syndrome were self-reported. Patient
P.II.5 (a 28-year-old North-American man carrying a deletion with a breakpoint centromeric to
OTULIN) reported pneumonia and recurrent skin and soft tissue infections. During his first year
of his life, the patient was admitted to the ICU for hemodynamic instability of unknown etiology.
At the age of 7 years and following a bilateral femoral osteotomy, the patient was admitted to the
ICU for respiratory insufficiency due to pneumonia. The pneumonia was progressive, and the
patient was placed on extracorporeal membrane oxygenation. Cultures, taken under treatment with
broad-spectrum antibiotics, remained negative. At the age of 10 years, the patient suffered from
an episode of unilateral mastitis requiring treatment with oral antibiotics. MRSA was isolated from
cultures. Superficial MRSA-infections recurred in the arm pits and groins until the age of 12 years.
At the age of 11 years, the patient was hospitalized for cellulitis of the wrist requiring treatment
with intravenous antibiotics. During this episode, cultures taken under treatment with antibiotics
remained negative. Patient Q.II.4 (a 28-year-old South-East-Asian woman man carrying a deletion
with a breakpoint centromeric to OTULIN) reported pneumonia. At the age of three months, she
was admitted for respiratory insufficiency of unknown etiology. The patient subsequently suffered
from multiple episodes of community-acquired pneumonia requiring treatment with oral
antibiotics. On several occasions, her clinical condition required admission to a regular ward for
observation. For as far as documented, sputum cultures had remained negative. The other
individuals with 5p- syndrome carrying a deletion with a breakpoint centromeric to OTULIN
(L.II.3, a 1-year-old French boy; M.II.3, a 1-year-old French boy; N.II.4, a 2-year-old girl of mixed
ancestry; O.II.3, a 21-year-old North-American woman) and those carrying a deletion with a
breakpoint telomeric OTULIN (R.II.4, a 2-year-old North-American girl; R.I.2, a 40-year-old
North-American man) reported no particular phenotype.

4
 Supplementary Materials and Methods
Whole-exome sequencing
An adaptor-ligated library was prepared with the TruSeq DNA Sample Prep Kit (Illumina).
Exome capture was performed with the SureSelect Human All Exon V5 Kit (Agilent
Technologies). Paired-end sequencing was performed on the HiSeq 2500 System (Illumina)
generating 100-base reads. Sequences were aligned with the GRCh37 build of the human genome
reference sequence with the Burrows-Wheeler Aligner (110). Downstream processing and variant
calling were performed with the Genome Analysis Toolkit (113), SAMtools (111), and Picard
tools (http://broadinstitute.github.io/picard/). Variants were subsequently annotated with in-house
annotation software. We validated genomic variants in patients and their relatives by amplifying
200 to 300 bp regions encompassing the mutation from gDNA samples with different sets of site-
specific primers. The amplicons were then sequenced with BigDye Terminator technology on an
ABI 3730 DNA sequencer. SnapGene (Version 3.1.4) was used for sequence analysis. Breakpoints
were determined by low-coverage whole-genome sequencing in individuals with 5p- syndrome,
as described elsewhere (101).

Generation and culture of induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) were generated from CD34+ cells (from patient
C.III.5) or from primary fibroblasts (from patient A.I.1) with the CytoTune-iPS 2.0 Sendai
Reprogramming Kit (Invitrogen). The manufacturer’s feeder-dependent protocol was followed to
reprogram CD34+ cells or primary fibroblasts, with the following exceptions: patient CD34+ cells
were isolated from cryopreserved PBMCs with the human CD34 MicroBead Kit (Miltenyi Biotec),
and 50,000 CD34+ cells or 50,000 primary fibroblasts were transduced with the standard kit MOIs.
CD34+ cells derived iPSCs from a healthy control and a patient with X-linked chronic
granulomatous disease (X-CGD) were kindly provided by Nico Lachmann and Alex Schambach,
respectively (Hannover, Germany) (106, 122). iPSCs were cultured on irradiated CF1 mouse
embryonic fibroblasts (Thermo Fisher Scientific) in iPSC medium (knockout DMEM containing
20% knockout serum replacement, 0.1 mM β-mercaptoethanol, 1 mM L-glutamine, 1%
penicillin/streptomycin, and 1% nonessential amino acids; all from Thermo Fisher Scientific)
supplemented with 20 ng/mL basic fibroblast growth factor (bFGF; PeproTech). iPSCs were
passaged every five to seven days, depending on colony size, with collagenase IV (Thermo Fisher
Scientific). Pluripotency was confirmed by an embryonic stem cell-like colony morphology,
expression of the pluripotency markers TRA1-60 and SSEA-4 and the expression of alkaline
phosphatase. All cell lines were karyotyped.

Hematopoietic differentiation of iPSCs into macrophages

iPSC-derived macrophages were generated with an embryoid body (EB)-based
hematopoietic differentiation protocol (122). In brief, iPSC colonies were disrupted with
collagenase IV, and EB formation was induced by culture in iPSC medium supplemented with 10
µM ROCK Inhibitor (Tocris) in six-well suspension plates incubated for five days on an orbital
shaker operating at 90 rpm, at 37°C, under an atmosphere containing 5% CO2. EBs were collected
manually, and myeloid cell complex formation was initiated in the presence of APEL2 medium
(Stem Cell Technologies) supplemented with 1% penicillin/streptomycin, 50 ng/mL macrophage
colony-stimulating factor (M-CSF; PeproTech) and 25 ng/mL interleukin-3 (PeproTech). After 14
days of incubation, macrophages were harvested once weekly from the supernatant and passed
through a filter with a 100 µM mesh. For further maturation, the harvested cells were cultured in

5
RPMI 1640 (Thermo Fisher Scientific) supplemented with 10% HI-FBS and 50 ng/mL M-CSF for
7-10 days. Successful differentiation was confirmed by the surface expression of CD11b, CD14
and CD163.

Phagocytosis assays and H2O2 production by iPSC-derived macrophages

For the assessment of phagocytosis, iPSC-derived macrophages were used to seed a 48-
well plate containing RPMI 1640 supplemented with 10% HI-FBS. After 24 hours, the medium
was replaced with live cell imaging solution (Thermo Fisher Scientific) and the cells were
incubated for 3 hours with fluorescently labeled S. aureus pHrodo red bioparticles (Thermo Fisher
Scientific) at 37°C, under an atmosphere containing 5% CO2. The cells were then washed
thoroughly in live cell imaging solution and bioparticle uptake was measured with a Gallios flow
cytometer (Beckman Coulter). NADPH oxidase activity in iPSC-derived macrophages was
assessed by measuring the extracellular release of H2O2 with the Amplex Red Hydrogen Peroxide
Kit (Thermo Fisher Scientific). In brief, cells were stimulated with 400 ng/mL phorbol 12-
myristate 13-acetate (PMA; Merck), and H2O2 release was quantified with a Victor X4 plate reader
(PerkinElmer).

Transcriptional analyses in primary dermal fibroblasts and PBMCs

Primary dermal fibroblasts were synchronized by incubation in DMEM supplemented with
1% HI-FBS for 24 hours before stimulation. RNA was extracted at steady state and after 6 hours
of stimulation with 20 ng/mL TNF (R&D Systems), with the RNeasyPlus Micro Kit (QIAGEN),
according to the manufacturer’s instructions. Freshly thawed PBMCs were left unstimulated or
were stimulated with 20 ng/mL TNF (R&D Systems), 10 ng/mL IL-1β (R&D Systems), HKSA
(MOI: 10; InvivoGen)), or 1 ng/mL LPS (InvivoGen) for 6 hours. Total RNA was extracted from
PBMCs with the Zymo Research RNA MicroPrep kit. RNA sequencing analysis was performed
with TruSeq Stranded mRNA (Illumina) and standard poly(A)-based methods for library
preparation. Paired-end sequencing, with a read length of 50 bp and 30-50 million reads per
sample, was performed with the NovaSeq S1 system (Illumina). We used STAR version 2.7.2b
(105) to align reads to the GRCh37.p13 genome. Reads were counted with HTseq (100), with the
parameter –nonunique=all and ensembl release 103 as the gene model. DESeq2 version 1.28.1
(112) was used for differential expression analyses. Genes with low levels of expression (fewer
than two samples with at least 1 count per million reads) were removed. Differential expression
was assessed with a multi-linear analysis, with mutation annotation as a factor. Gene set
enrichment analyses were performed on Hallmark gene sets with GSEA software (version 4.1.0),
with ranking of the log2 fold-change in expression relative to the controls as the input. The
expression of OTULIN, CAV1, CAV2, and GSN in resting and TNF-stimulated cells were
represented relative to the mean values for conditional controls and merged per gene for each
individual.

Single-cell RNA sequencing on PBMCs

Single-cell RNA sequencing was performed on PBMCs obtained from four patients with
OTULIN haploinsufficiency (three of whom carried heterozygous mutations of OTULIN, plus one
5p- syndrome patient) and four healthy controls. The samples were prepared as described
elsewhere (96). Cells were resuspended at a density of 1000 cells/μL in PBS + 0.04% BSA and
loaded onto a 10X Genomics Chromium chip for single-cell capture. Reverse transcription and
library preparation were performed with Chromium Single Cell 3′ Reagent Kits (v3) in accordance

6
with the manufacturer’s guidelines, and library quality was assessed with a Bioanalyzer DNA chip.
Each library was sequenced on two lanes of a NovaSeq S2 sequencer in a 28x94x8 paired-end
configuration. We aligned the single-cell RNA-seq data and generated count matrices with the
cellranger count function of cellranger 3.1.0 (120), with the default parameters. The gene count
data were further analyzed as previously described (123), but with minor modifications. Briefly,
low-quality cells were excluded by manual thresholding based on the number of features detected
and the percentage of mitochondrial genes. Graph-based clustering was performed after batch-
correction with Harmony (121). Clusters were identified manually from the patterns of expression
for lineage marker genes. Two TotalSeq datasets obtained from the 10X Genomics website were
also included in the analysis, to facilitate cluster identification. Clusters corresponding to platelets
and erythrocytes were excluded from subsequent analyses. Cluster-wise pseudobulk differential
expression analysis was conducted with NOISeq (124). The expression patterns of representative
DE genes were visualized via uniform manifold approximation and projection (UMAP). All
analyses were conducted in R v.4 (https://www.r-project.org/).

Immunofluorescence deubiquitination and competition assays

For immunofluorescence deubiquitination assays, cells were incubated for 10 minutes at
room temperature in deubiquitinase buffer containing 50 mM Tris pH 7.5, 50 mM NaCl, 2 mM
DTT, 5 mM MgCl2 and 0.1% BSA. Cells were then treated for 45 minutes at 37°C with 1 μM
OTULIN (WT or C129S) or 6 μM vOTU in deubiquitinase buffer. Mock-treated cells were
incubated with deubiquitinase buffer alone. After four washes with PBS, cells were incubated with
1% BSA in PBS for 30 minutes. An indirect immunofluorescence assay was then performed with
the anti-M1-Ub antibody, as described in the Materials and Methods. The recombinant vOTU
deubiquitinase (OTU domain of the Crimean Congo hemorrhagic fever virus L1 protein) was
produced as a GST fusion protein from a bacterial expression vector encoding GST-vOTU
CCHFV-L (1–169). Recombinant OTULIN His-tagged fusion proteins were produced from
bacterial expression vectors encoding OTULIN-WT and OTULIN-C129A (wild-type and the
catalytic-dead mutant C129A) (30). For immunofluorescence competition assays with free
polyubiquitin chains, incubation with the primary antibody was performed in the presence of 20
μg/mL tetra-M1 or K63-Ub chains (R&D Systems).

Global quantitative proteomics in primary dermal fibroblasts with isobaric labels

PDFs were synchronized by incubation in DMEM containing 1% HI-FBS for 24 hours and
were then harvested by trypsin treatment. Cell pellets were lysed in WCL buffer C (Table S4),
with sonication to clear the supernatants. Proteins were precipitated from the lysate in
chloroform/water/methanol (118) and pellets were dissolved in 8 M urea, 50 mM
triethylammonium bicarbonate (TEAB). Reduction and alkylation were performed with
dithiothreitol and iodoacetamide, respectively. Proteins were digested with LysC (2% w/w
overnight at room temperature) and trypsin (2% w/w for 6 hours at room temperature). We then
withdrew 110 µg peptides from each sample for purification on Oasis HLB cartridges (Waters)
according to the manufacturer’s instructions. Eluates were dried by vacuum centrifugation and
dissolved in 100 mM TEAB. Samples were divided into two TMT-labeling groups. We removed
a small proportion from each sample for combination to form a normalizer sample for comparison
between the two TMT groups. The purified peptides were labeled with 400 µg TMT-10 plex
reagent dissolved in anhydrous acetonitrile and the normalizer sample was labeled with the 11th
plex reagent. Labeling was allowed to proceed for 1 hour and was quenched by adding 5%

7
hydroxylamine. We collected a small aliquot from each sample. These aliquots were combined
according to their labeling group and analyzed by LC-MS/MS to assess labeling efficiency and
stoichiometry. Samples were pooled and amounts were adjusted according to the labeling
assessment. Peptides were fractionated by basic-pH reversed-phase HPLC with a Dionex 3000
Ultimate loading pump equipped with a 2.1*150 mm 3.5 µm Xbridge C18 column (Waters).
Solvent A consisted of 10 mM ammonium hydroxide (Sigma-Aldrich) in water, pH 10, and solvent
B consisted of 10 mM ammonium hydroxide, 90% acetonitrile (ACN) in water, pH 10. Peptides
were fractionated across a 60-minute gradient and the collected fractions were concatenated to
yield a total of 24 fractions. For the next step, solvent A was 0.1% formic acid in water and solvent
B was 0.1% formic acid, 80% acetonitrile (ACN) in water. All LC-MS solvents were of HPLC
grade and were purchased from Sigma. Peptide fractions were separated by HPLC on a Dionex
3000 Ultimate equipped with a NCS3500RS nano- and microflow pump (Dionex). Peptides were
loaded onto a 100 µm x 20 mm Acclaim PepMap C18 trap column (Thermo Scientific), with
elution at a rate of 3 µL/min. Separation was achieved with a 75 µm x 120 mm pulled emitter
nanocolumn (Nikkyo Technos). The proportion of solvent B increased from 1% to 5 % over 6
minutes, then to 34% over 70 minutes, and then increased sharply, over a period of one minute, to
90%, a proportion at which it was maintained for 12 minutes. Peptides were analyzed with a Q-
Exactive HF mass spectrometer (Thermo Fisher Scientific). Data were recorded in positive mode
with Top 20 DDA acquisition. Resolution was set to 60,000 for both MS1 and MS2. AGC targets
of 3e6 (MS1) and 2e5 (MS2) were applied. Raw data were analyzed with MaxQuant version
1.6.6.0 (102). Spectra were used to query the human proteome (downloaded from uniprot.org on
February 12, 2019, 73931 sequences) with a false discovery rate (FDR) of 1%. Protein
quantification results were processed further with the Perseus software platform (115). Values
were log2-transformed and samples were normalized against both the median, to compensate for
variation of the amounts within runs, and the normalizer channel, for comparisons between runs.
Significance was assessed in an FDR-corrected t-test. Gene set enrichment analyses were
performed on Hallmark gene sets with GSEA software (version 4.1.0), with log2-fold changes in
expression relative to the controls as the input. Highest-confidence in silico analyses of functional
and physical protein-protein interactions were performed with the STRING database (version
11.5).
Fractions found to contain OTULIN peptides were analyzed along with the corresponding
fraction from the other run, by a parallel reaction monitoring (PRM) method, allowing
quantification of the same peptides across both runs. The masses of four OTULIN peptides
identified in large-scale analysis (AIELYNDK, DTSNDPGQLLR, EAAATAR and
VRGDNYCALR) were added to an inclusion list and analyzed with a Fusion Lumos Tribrid mass
spectrometer (Thermo Scientific) using both PRM and DDA within the same cycle. Separation
was achieved with an Easy nLC-1200 (Thermo Fisher Scientific) system equipped with a 250 µm
x 0.75 mm Easyspray column (Thermo Fisher Scientific). Data were processed with Proteome
Discoverer v.2.4 (Thermo Fisher Scientific), with the same database settings as described above.
Signals from OTULIN peptides were first normalized against the median for all identified proteins
within each channel, and then against the normalizer group within each TMT-group.

Targeted proteomic analyses on GlyGly-tagged peptides in purified caveolin-1

High-MW and low-MW bands obtained by gel electrophoresis of the immunopurified
caveolin-1, corresponding to caveolin-1, as demonstrated by western blotting, were excised from
the gel with a scalpel. The gel fragments were destained, reduced, alkylated and digested, as

8
described elsewhere (128), except that acrylamide (incubation with 55 mM in 100 mM ammonium
bicarbonate for 30 minutes at room temperature in the dark) was used for alkylation instead of
iodoacetamide. Peptides were obtained through three rounds of extraction in 70% acetonitrile, 5%
formic acid in water, and were dried by vacuum centrifugation. Eluates from in-solution
immunopurification were mixed with ice-cold acetone and incubated overnight at -20°C. The
resulting pellets were dissolved in 8 M urea, 50 mM TEAB and 10 mM DTT. The reduction
reaction was allowed to proceed for 1 hour at room temperature. Cysteines were alkylated by
incubation with 20 mM acrylamide for 30 minutes at room temperature in the dark, and DTT was
then added to quench the reaction. Peptides were purified with C18 micropurification tips
produced in-house and dried by vacuum centrifugation. Samples were analyzed by two rounds of
mass spectrometry on a Q-Exactive HF mass spectrometer operating in combined DDA/PRM
mode, making it possible both to identify unknown proteins and to perform targeted quantification
for several ubiquitin- and caveolin-derived tryptic peptides with GlyGly tags [+114 Da]. LC
instrumentation and solvent conditions were identical to those described above, except for the use
of gradients of 50- or 90-minutes duration. The targeted peptides for caveolin-1 were (residues
modified with a GlyGly tag underlined; residues alkylated by acrylamide in italics and underlined):
SGGKYVDSEGHLYTVPIR; YVDSEGHLYTVPIREQGNIYKPNNK; EQGNIYKPNNK; EQ-
GNIYKPNNKAMADELSEK; EQGNIYKPNNKAMADELSEK; AMADELSEKQVYDAHTK;
QVYDAHTKEIDLVNR; DPKHLNDDVVK; ASFTTFTVTKYWFYR; VYSIYVHTVCDPLFE-
AVGKIFSNVR; INLQKEI. The targeted peptides for ubiquitin were (residues modified with a
GlyGly tag underlined): MQIFVK; ESTLHLVLR; MQIFVKTLTGK; TLTGKTITLEVEPSDT-
IENVK; TLTGKTITLEVEPSDTIENVKAK; TITLEVEPSDTIENVKAK; TITLEVEPSDTIEN-
VKAKIQDK; AKIQDK; IQDKEGIPPDQQR; LIFAGKQLEDGR; TLSDYNIQKESTLHLVLR.
Spectra were used as queries against the human proteome, with an FDR of 1%, with the MASCOT
search engine and Proteome Discoverer v. 1.4 (Thermo Fisher Scientific). Proteins for which at
least two peptide species were identified, and for which the abundance in caveolin-1 in-solution
immunopurification eluates was at least 1000 times higher than that in isotype control eluates,
were retained for comparisons between the patient and healthy controls. The peak areas for the
targeted peptides were extracted with Skyline v. 20.2.0.343 (127). All matching MS2 ions were
summed and used for quantification without smoothing. Peak areas were normalized against the
top3 signal for caveolin-1 for each sample.

M1-Ub quantification and M1-Ub-bound peptide identification

TUBE protein samples were run on a 12% acrylamide SDS-PAGE gel with a short
migration and were stained with silver nitrate. The band corresponding to the TUBE protein was
removed to prevent saturation effects. The rest of the sample was treated as a single band and was
subjected to in-gel digestion. The gel pieces were sliced into 1 mm3 cubes and destained by
washing twice with a freshly prepared solution containing 15 mM K3[Fe(CN)6] and 50 mM
Na2S2O3. The proteins were then reduced with 10 mM DTT in 50 mM NH4HCO3 for 30 minutes
at 56°C and alkylated with 50 mM N Ethylmaleimide (NEM) in PBS for 30 minutes at room
temperature in the dark. Proteins were digested by overnight incubation with 300 ng trypsin per
sample, at 37°C, and the tryptic peptides were collected. The gel pieces were washed twice in 60
% acetonitrile (ACN)/0.1 % trifluoroacetic acid for 10 min in an ultrasonic bath. The extracted
peptides were dried in a rapid vacuum dryer and resuspended in 20 µL 2% ACN/0.1% formic acid
(FA). Peptides were diluted with 50 µL 0.5% acetic acid (HAc) and desalted on stage tips with
three layers of Empore SPE Disks C18 (Sigma Aldrich), with the DigestProMSi robot (Intavis).

9
They were washed with 0.5% HAc and eluted with 80% ACN/0.5% HAc, dried in a rapid vacuum
dryer and resuspended in 20 µL 2% ACN/0.1% FA. All samples were stored at - 20°C until MS
analysis. The synthetic heavy M1 ubiquitin peptide GGMQIFV[13C515N1]K was purchased from
Sigma (The Woodlands, TX, USA). This AQUA peptide was dissolved at a concentration of 100
fmol/µL in 2% ACN/0.1% FA and purity was monitored by ESI-MS and LC-MS/MS on a
timsTOF Pro (Bruker), by trapped ion mobility spectrometry in DDA PASEF mode (Parallel
Accumulation–Serial Fragmentation), as described below. Both the oxidized and non-oxidized
forms of the peptide were detected. The oxidized peptide chromatogram contained a double peak,
as previously observed for some ubiquitin peptides (104). Each sample was incubated overnight
with 0.05% H2O2 to induce complete methionine oxidation. To construct a calibration curve, a mix
of total protein digests from two patients (Mix 1) was spiked with the M1 AQUA peptide at
concentrations ranging from 3 amol/µL to 500 fmol/µL. To adjust the slope of the calibration curve
for TUBE-enriched protein extracts, a second mix was prepared with equal amounts of each
TUBE-enriched sample (Mix2). Next, Mix 2 was spiked with three different dilutions of the M1
AQUA peptide: 125 amol/µL, 625 amol/µL and 3.125 fmol/µL. For endogenous M1 peptide
quantification, the M1 AQUA peptide was used to spike each individual sample at 125 amol/µL
as an internal standard. Peptide mixtures were analyzed with a nanoElute UHPLC (Bruker)
coupled to a timsTOF Pro mass spectrometer (Bruker). Peptides were separated on an RP-C18
Odyssey (25 cm, 75 μm i.d., 120 Å, 1.6 µm particle size, IonOpticks) analytical column at a flow
rate of 400 nL/min, at 50°C, with mobile phases A (FA 0.1%) and B (ACN 99.9%/FA 0.1%). The
elution gradient was as follows: 3% to 15 % B in 17 min then 17% to 23% B in 7 min, 23% to
32% B in 5 min and 32% to 85% in 3 min. MS acquisition was performed in DDA mode with
PASEF as described elsewhere (125). Raw data were processed with Data Analysis 5.1 (Bruker)
to generate extracted ion chromatograms of the peaks at 448.2390 ± 0.005 m/z for the endogenous
M1-Ub peptide and 451.24 ± 0.005 m/z for the heavy AQUA M1 peptide. Each point on the
calibration curve for the Mix1 sample resulted from three injections. Peak intensities were
extracted manually for each sample. A standard curve was generated from the log-transformed
mean area under the curve (AUC) values versus M1-Ub AQUA peptide abundance. This
calibration curve was used to determine the lower limit of detection (LLOD) and the lower limit
of quantification (LLOQ). The LLOD was determined as the [(mean AUC (Blank) + 3 x standard
deviation (Blank)) /slope] and the LLOQ was determined as the [mean AUC (Blank) + 10 x
standard deviation (Blank)) /slope] where the blank corresponds to the sample mix with no AQUA
peptide. The LLOD was 130 amol, and the LLOQ was 434 amol. The Mix2 sample was used to
validate the linearity of M1 AQUA peptide detection in the selected range in TUBE-enriched
protein extracts and to adjust the slope of the calibration curve. All R² values were > 0.99. The
abundance of the endogenous M1-Ub peptide was calculated from the AUC of the AQUA M1-Ub
peptide used to spike each sample.
For protein identification, X!tandem (version 2017.2.1.4) was used with the X!tandem
pipeline (version XTPCPP 0.4.3) and the Human UniProt database (version 20201007, 20614
entries) as a reference (126). The applied permissive parameters were as follows: i) 3 missed
cleavages; ii) 25 ppm for MS and MS² tolerance; iii) fixed modification: Cys-NEM and Met-Ox;
and iv) variable modifications: Lys-KK and acetyl N-term. Proteins were identified with at least 1
unique peptide and when both the peptides and corresponding proteins had an FDR < 1%. For
selection of peptides potentially bound to M1-Ub, the following criteria were used: i) detection of
a peptide in >1 individual, ii) for which the number of spectra detected was >1 in at least one
individual, iii) combined with the absence of spectra in the HOIL1-deficient patient. Highest-

10
confidence in silico analyses of functional and physical protein-protein interactions were
subsequently performed with the STRING database (version 11.5).

Bacterial culture supernatant and recombinant toxins

Staphylococcus aureus strain USA300 clone LAC, an epidemic clone of methicillin-
resistant S. aureus (103, 114), was grown in tryptic soy broth. Crude overnight cultures were
centrifuged, and the supernatant was cleared by passage through a filter with 0.2 μm pores. α-
toxin-Cys was produced and labeled with AF647 as described elsewhere (117). α-toxin was
cloned, expressed, and purified from S. aureus as previously described (109). Aerolysin from
Aeromonas hydrophilia ATCC 7966 (PubMed gene ID 4490401) and streptolysin O (SLO) from
Streptococcus pyogenes GAS M1 (PubMed gene ID 900490) were ordered as gBlocks from
Integrated DNA Technologies. Aerolysin was cloned as proaerolysin with a C-terminal 6-histidine
tag through restriction digestion with NcoI and XhoI and ligation into the pET28a expression
vector (119). SLO was cloned with an N-terminal 6-histidine tag through restriction digestion with
NheI and XhoI and ligation into pET28a (116). The resulting plasmids were used to transform E.
coli DH5α competent cells, with transformants selected on the basis of kanamycin resistance (50
µg/mL), and confirmed by colony PCR and Sanger sequencing (Genewiz). The purified plasmids
were then used to transform the E. coli T7 Express lysY/lq strain (New England Biolabs). The
expression strains were grown overnight at 37°C, with shaking at 180 rpm, in Luria-Bertani (LB)
broth supplemented with kanamycin (50 µg/mL). The following day, the overnight cultures were
subcultured at a 1:20 dilution in LB supplemented with kanamycin (50 µg/mL) and were grown
to an OD600 of 0.5-0.6 (~1.5 hours) at 37°C, with shaking at 180 rpm. The expression of
proaerolysin and SLO was induced by incubation with 1 mM IPTG in LB broth supplemented with
kanamycin (50 µg/mL) for 16 hours at 16°C, with shaking at 220 rpm. The bacteria were then
pelleted by centrifugation at 10,000 rpm, for 15 min at 4°C then the bacterial pellets were frozen
at -20°C. The bacterial pellets were thawed on ice and resuspended in lysis buffer (50 mM
Na2HPO4, 500 mM NaCl, 10 mM imidazole, 1X protease inhibitor (Thermo Fisher Scientific),
140 U/mL Benozonase, and 2 mM MgCl2). The bacterial cells were lysed by sonication for 2
minutes on ice at 65% amplitude, and were then treated with BugBuster for 30 min at room
temperature, with shaking, before being placed on ice for 30 min. The lysates were centrifuged at
10,000 rpm, for 30 minutes at 4°C and the supernatant (cleared lysate) was filtered through a filter
with 0.22 μm pores (Corning). The cleared lysate was then diluted 1:1 into 50 mM Na2HPO4 with
10 mM imidazole, 1X protease inhibitor, and 10% glycerol and incubated with equilibrated
nitrilotriacetic acid (Ni-NTA) agarose resin (Qiagen) for 1 hour at 4°C, with shaking. The cleared
lysate-Ni-NTA resin mixture was then applied to a glass column by gravity filtration, and the ni-
NTA-bound proteins were washed with 20 mM Na2HPO4, 250 mM NaCl with 10 mM imidazole,
followed by 20 mM Na2HPO4, 250 mM NaCl with 25 mM imidazole. The ni-NTA-bound proteins
were then eluted in 500 mM imidazole. The purified proteins were dialyzed against 20 mM
Na2HPO4, 200 mM NaCl plus 10% glycerol and passed through a filter with 0.22 μm pores before
storage at −80°C. Protein concentration was determined on the basis of absorbance at 280 nm with
a Nanodrop spectrometer (Thermo Fisher Scientific) and Beer-Lambert’s equation. We subjected
1 μg of the purified proteins to SDS-PAGE at 90 V for 120 minutes, and the gel was then stained
with Coomassie Brilliant Blue to confirm purity. The cytotoxic activity of purified aerolysin and
SLO was confirmed with primary human neutrophils. Furin cleavage of proaerolysin was not
required for aerolysin cytotoxicity in primary human neutrophils.

11
 Mice
All experiments involving animals were reviewed and approved by the Institutional Animal
Care and Use Committee of New York University and were performed according to guidelines
from the National Institutes of Health (NIH), the Animal Welfare Act, and the U.S. Federal Law.
C57BL/6 Otulinflox mice were provided by David Komander and Rune B. Damgaard (MRC
Laboratory of Molecular Biology, Cambridge, UK) (30), re-derived and bred in-house at NYU
Grossman School of Medicine. Otulinflox mice were crossed with creERT2 (#008463) mice
purchased from The Jackson Laboratory. CreERT2-Otulinflox mice were treated with tamoxifen
(#T5648-1G) by i.p. injection on three consecutive days to ablate Otulin, as previously described
(30). Otulin ablation was confirmed by PCR. Mice were maintained under specific pathogen-free
conditions, and age-matched mice were used for experiments at the age of 8-10 weeks.

Anti-α-toxin immunoglobulins
We assessed the capacity of plasma to neutralize microbial toxins as described elsewhere
(61). Briefly, rabbit erythrocytes (Innovative Research) were washed and their density was
adjusted to 1.5 x 108 cells/mL in PBS supplemented with 0.5% BSA. Toxins were incubated for
30 minutes at room temperature with diluted plasma. Rabbit erythrocytes were added and the
mixture was incubated for 1 hour at 37°C, under an atmosphere containing 5% CO2, with shaking
at 600 rpm. The supernatant was cleared by brief centrifugation and its OD415 was measured on a
spectrophotometer. Neutralizing capacity is expressed relative to a control sample not treated with
toxin. Anti-α-toxin immunoglobulins were quantified by ELISA; as described elsewhere (61).
Briefly, a microtiter plate was coated with 3 μg/mL α-toxin in carbonate/bicarbonate buffer and
incubated at 4°C overnight. The plate was washed and blocked, and plasma dilutions were applied.
The plate was then incubated for 1 hour at room temperature. It was washed, and peroxidase-
conjugated anti-human-IgG, anti-human-IgA, or anti-human-IgM (all EMD Millipore) was
applied. The plate was then incubated for another hour at room temperature. The plate was
thoroughly washed, 3,3′,5,5′-tetramethylbenzidine substrate was added and the plate was
incubated for 10 minutes at room temperature. The reaction was stopped by adding sulfuric acid,
and the plate was read on a spectrophotometer at 450 nm with correction at 540 nm.

12
13
14
 Figure S1. OTULIN haploinsufficiency and its molecular characterization. Related to
Figure 1. (A) Clinical images of necrotic sequelae triggered by S. aureus infection in the patients.
Bilateral pneumonia with progressive consolidations and alveolar edema on chest X-rays of patient
A.II.5 at the day of admission (day 0; left image) and at the day before demise (day 4; right image).
Areas of grafted skin on the right arm (left image) and waist (right image) following recurrent
episodes of necrotizing cellulitis in patient B.III.5. Postsurgical scars of the abdominal wall (left
image) and skin grafts in the neck (right image) following abscess formation and necrotizing
fasciitis, respectively, in patient C.III.5. (B) Clinical images of necrotic sequelae triggered by other
etiologies. Computed tomography (CT) images of different episodes of necrotizing pneumonia in
patient D.II.5 (stars indicate consolidations; arrows indicate necrotic cavities). Necrotic lesion of
the skin surrounding a post-surgical wound in patient E.II.5. (C) Schematic overview of the
OTULIN variants found in the patients. PIM: PUB-interacting motif; OTU: ovarian tumor
deubiquitinase domain. (D) The OTULIN ƒ-parameter indicates negative selection pressure. (E)
The OTULIN consensus score for negative selection (CoNeS) is in the range of those for inborn
errors of immunity with both autosomal dominant (AD) and autosomal recessive (AR) patterns of
inheritance. (F) Population genetics of OTULIN, based on the minor allele frequencies (MAF)
reported in the gnomAD database plotted against combined annotation-dependent depletion
(CADD) scores. LOF: loss of function. (G) Expression of OTULIN and its gene product in
transiently transfected HEK293T cells, as detected by RT-qPCR (N=4-5, mean ±SD) and western
blotting. (H) Expression of truncated variants with a C-terminal in-frame FLAG-tag. (I) Linear
deubiquitinase capacity of OTULIN variants in HEK293T cells transiently cotransfected with
LUBAC components and NEMO. The lanes were reorganized post-acquisition to facilitate visual
comparison with other figure panels. (J) NF-κB inhibitory capacity of OTULIN variants assessed
with a luciferase reporter system in transiently transfected HEK293T cells stimulated with TNF
(N=4-5, mean ±SD). (K) NF-κB inhibitory capacity of OTULIN variants, as assessed with a
luciferase reporter system in HEK293T cells transiently cotransfected with wild-type OTULIN
and stimulated with TNF (N=3-4, mean ±SD). Numbers represent ratios of empty vector (EV) and
OTULIN-encoding vectors. (L) Breakpoints in study participants with 5p- syndrome, as
determined by low-coverage whole-genome sequencing.

15
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17
 Figure S2. OTULIN haploinsufficiency does not impair the hematopoietic immune
system. Related to Figure 2. (A) Expression of OTULIN in monocytes. (B) Individual uniform
manifold approximation and projection (UMAP) plots of PBMCs from patients with autosomal
dominant (AD) OTULIN deficiency and healthy controls, as assessed by cytometry by time-of-
flight (CyTOF). (C) Abundances of the various PBMC subsets for the patients; comparison with
adult (A) and pediatric (P) controls, as assessed by CyTOF. (D) Lineage marker expression profiles
for the classification of leukocyte subsets, as assessed by CyTOF. (E) Gene-specific enrichment
analysis of pathways contributing to the separation of transcriptional profiles at baseline between
PBMCs from patients and PBMCs from healthy controls. (F) Secretion of TNF, IL-6, and IL-1β
by PBMCs at baseline and following stimulation (median). (G) Differential expression analysis
for bulk RNA sequencing data for PBMCs from the patients combined with single-cell RNA
sequencing data for their CD14+ monocytes, in comparison with those from healthy controls. Black
dots represent genes identified as significantly upregulated or downregulated in both the bulk RNA
sequencing data and the single-cell RNA sequencing data for CD14+ monocytes. (H) UMAP plots
of single-cell RNA sequencing data for PBMCs from patients with OTULIN haploinsufficiency
and healthy controls, with expression data for two representative genes differentially expressed at
baseline in both the bulk RNA sequencing data and the single-cell RNA sequencing data for CD14+
monocytes. (I) Intracellular cytokine staining for TNF and IL-1β in CD14+ monocytes at baseline
and following stimulation (median). (J) Phosphorylated P65 levels in CD14+ monocytes at
baseline and following stimulation, relative to those in a healthy donor internal control, as
determined by flow cytometry (median). (K) Oxidative burst in induced pluripotent stem cell
(iPSC)-derived macrophages (mean ±SD, N=3-6). (L) Phagocytic capacity of iPSC-derived
macrophages for S. aureus (mean ±SD, N=3).

18
19
 Figure S3. OTULIN gene dosage-dependent accumulation of M1-Ub in fibroblasts.
Related to Figure 3. (A) OTULIN expression in primary dermal fibroblasts (PDFs), as determined
by RNA sequencing and expressed relative to the mean value for controls (median, N=2). (B)
OTULIN expression in PDFs, as determined by RT-qPCR, expressed relative to the mean for the
controls (median, N=3). (C) Quantification of aggregates containing M1-Ub in immortalized
fibroblasts, as assessed by immunohistochemistry. (D) Aggregates containing M1-Ub in
immortalized fibroblasts treated with recombinant vOTU, wild-type human OTULIN, or
catalytically dead OTULIN (p.C129A). (E) Quantification of aggregates containing M1-Ub in
immortalized fibroblasts transduced with an empty virus or with wild-type OTULIN. For the cell
line with a biallelic OTULIN deficiency, comparative analyses were performed on cells with high
and low levels of exogenous OTULIN expression after viral transduction with wild-type OTULIN.
(F) NF-κB signaling in immortalized fibroblasts upon stimulation with TNF. (G) Cytokine
secretion in PDFs, as detected by ELISA (datapoints indicate the mean of N=3 replicates per
patient, the median per group). (H) Transcriptional response of PDFs following 6 h of stimulation
with TNF, expressed as the log2-fold changes (FC) relative to the mean value for unstimulated
controls. Genes satisfying the threshold for statistical significance upon stimulation with TNF
relative to baseline (N=3,344), as detected by RNAseq, are shown. (I) Viability of PDFs after
stimulation with a combination of TNF and BV6 (median). The statistical significance of
differences was calculated by ANOVA with Dunnett post hoc correction for multiple comparisons.

20
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22
 Figure S4. OTULIN-dependent accumulation of caveolin-1 in fibroblasts. Related to
Figure 4. (A) Gene-specific enrichment analysis (GSEA) of the transcriptional profile of
unstimulated primary dermal fibroblasts (PDFs), with Hallmark or random gene sets, with log2-
fold changes (FC) for the specific groups relative to the mean in controls for all genes used as an
input for ranking. (B) Overlap between the gene sets displaying enrichment in the patients. (C)
GSEA on the proteomic profiles of unstimulated PDFs, with Hallmark or random gene sets, with
log2FCs relative to the mean values in controls for all detected proteins used as an input for ranking.
(D) Identification of proteins satisfying the threshold for statistical significance for differential
abundance in various genotypes. (E) Abundance of caveolin-2 in whole-cell lysates (WCLs) from
PDFs. Caveolin-2 intensities relative to β-actin intensities, normalized against the mean value for
healthy controls (datapoints indicate the mean of N=2 replicates per patient, the median per group).
(F) Abundance of gelsolin in WCLs of PDFs. Gelsolin intensities relative to β-actin intensities,
normalized against the mean value for healthy controls (datapoints indicate the mean of N=2
replicates per patient, the median per group). (G) Peptide abundance analysis by LC-MS/MS
following the immunopurification of caveolin-1 in WCLs from PDFs. Dotted lines indicate
detection thresholds; the dashed line indicates a hypothetical correlation coefficient of one. (H)
Caveolin-1 levels in WCLs from monocytes relative to PDFs. (I) Absence of accumulation of the
high-MW caveolin-1-containing complex in PDF-WCLs from a 5p- syndrome patient with a
breakpoint telomeric to OTULIN. (J) Accumulation of high-molecular weight (MW) caveolin-1-
containing complexes in WCLs from isogenic PDFs treated with an sgRNA pool targeting
OTULIN (sgOTU) or a non-targeting control sgRNA (sgNTC). The statistical significance of
differences was assessed in Student’s t-tests.

23
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 Figure S5. Polyubiquitination of caveolin-1. Related to Figure 5. (A) Expression of
CAV1, CAV2, and GSN in primary dermal fibroblasts (PDFs), as determined by RNA sequencing,
expressed relative to the mean value for controls (median, N=2). (B) Transcriptional and proteomic
profile of unstimulated PDFs, analyzed by gene ontology for specific biological pathways. (C)
Caveolin-1 (CAV1) or isotype control (ISO) immunopurification products (IPs) from whole cell
lysates (WCLs) of PDFs subjected to immunoblotting for caveolin-1, total ubiquitin, K63-Ub, and
M1-Ub on independent blots. (D) Analysis of K48-Ub and unmodified ubiquitin on caveolin-1 by
LC-MS/MS in caveolin-1 IPs from PDF WCLs. Caveolin-1 was analyzed in solution, in the high-
molecular weight (MW) complex fraction, and in the monomeric (Mono) fraction. (E) High-MW
K63-Ub complex in recombinant deubiquitinase-treated purified caveolin-1 from PDFs. (F) High-
MW K63-Ub complex in purified caveolin-1 from PDFs after rescue with CYLD and/or OTULIN.
(G) Colocalization of caveolin-1 with M1-Ub and K63-Ub aggregates in immortalized fibroblasts,
as assessed by immunohistochemistry. (H) STRING analysis of the cluster of proteins identified
by AQUA-MS/MS in TUBE pull-downs from immortalized fibroblasts. (I) Western blot analysis
of caveolin-1, CYLD, and HOIP bound to linear ubiquitin, as detected in TUBE pull-downs from
immortalized fibroblasts. (J) Intensities of CYLD and HOIP bound to M1-Ub, as detected in
TUBE pull-downs from immortalized fibroblasts relative to intensities of total CYLD and HOIP,
respectively. * high-MW caveolin-1-containing complex; # rabbit IgG. ND: not detected.

27
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 Figure S6. OTULIN haploinsufficiency impairs intrinsic immunity to α-toxin in
fibroblasts. Related to Figure 6. (A) Images from a representative experiment, acquired after 30
minutes of incubation of primary dermal fibroblasts (PDFs) in the absence or presence of α-toxin,
used as input for the quantification of colocalization. (B) Quantification of the colocalization of α-
toxin and ADAM10, α-toxin and caveolin-1, and of ADAM10 and caveolin-1 in PDFs, as assessed
by confocal microscopy (datapoints indicate the mean of N=3 per patient, the median per group).
(C) Binding of α-toxin in PDFs, as assessed by flow cytometry, relative to the mean value for
controls (datapoints indicate the mean of N=3 per patient, the median per group). (D) ADAM10
surface expression on PDFs, as detected by flow cytometry, relative to a healthy donor internal
control (datapoints indicate the mean of N=1-3 replicates per patient, the median per group). (E)
ADAM10 levels in whole-cell lysates from PDFs. (F) ADAM10 surface expression following the
addition of α-toxin relative to baseline, with detection by flow cytometry (datapoints indicate the
mean of N=3 replicates per patient, the median per group). (G) Viability of PDFs following 2.5 h
of incubation with culture supernatant from S. aureus pretreated with monoclonal antibodies
neutralizing α-toxin (MEDI-4893, ASN-F5) or against 2,4-dinitrophenol (DNP) (datapoints
indicate the mean of N=3 per patient, the median per group). (H) Maximal half-effective
concentrations (EC50) in PDFs after incubation for 24 h with recombinant toxins (N=2-4 per group,
mean ±SD per group). EC50 values were calculated in nonlinear regression analyses. (I) Viability
of PDFs from a patient with 5p- syndrome with a breakpoint telomeric to OTULIN (WT/WT(5p-
)) relative to those from a healthy control (WT/WT), following 24 h of incubation with
recombinant α-toxin (N=3, mean ±SD). (J) Simple linear regression analysis of cell surface
metalloprotease activation (Figure 3G) versus ADAM10 expression (Figure 3C) following α-toxin
binding to PDFs. (K) Viability of PDFs after treatment with an ADAM10-inhibitor or carrier, or
with cyclodextrin followed by 24 h of incubation with α-toxin (datapoints indicate the mean of
N=2 per patient, the median per group). (L) Viability in OTULIN-deficient PDFs treated with an
sgRNA pool targeting CAV1 (sgCAV1), CAV2 (sgCAV2), or a non-targeting control sgRNA
(sgNTC) (N=3, mean ±SD). (M) Viability of peripheral blood mononuclear cells (PBMCs) (N=1-
3 per patient, mean ±SD) and induced pluripotent stem cell (iPSC)-derived macrophages (N=3 per
patient, mean ±SD) after incubation for 2 h (PBMCs) or 24 h (iPSC-derived macrophages) with
recombinant α-toxin. (N) Viability of mouse PDFs after 24 h of incubation with recombinant α-
toxin (N=3, mean ±SD). Statistical significance was assessed by ANOVA with Dunnett post hoc
correction for multiple comparisons.

30
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 Figure S7. α-toxin-neutralizing antibodies rescue OTULIN haploinsufficiency.
Related to Figure 7. (A) The capacity of plasma to neutralize the hemolytic activity of α-toxin
(20 ng/mL), pneumolysin (PLY, 0.67 ng/mL), and streptolysin O (SLO, 42 ng/mL) was assessed
with rabbit erythrocytes. (B) Contribution of IgG to the neutralizing capacity of plasma. (C) Anti-
α-toxin immunoglobulin levels in plasma, as determined by ELISA. (D) Contribution of IgG to
anti-α-toxin immunoglobulin levels. (E) Total-IgG levels in the samples tested, with detection by
ELISA. Capacity of plasma to neutralize the hemolytic activity of α-toxin at a dilution of 1:300,
corrected for total IgG levels. Anti-α-toxin IgG levels, corrected for total IgG levels. The statistical
significance of differences was determined by ANOVA with Dunnett post hoc correction for
multiple comparisons.

33
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